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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 2 655-662
Copyright © 2003 by The Endocrine Society

Ovarian Steroids Differentially Modulate the Gene Expression of Gonadotropin-Releasing Hormone Neuronal Subtypes in the Ovariectomized Cynomolgus Monkey

Sally J. Krajewski, Ty W. Abel, Mary Lou Voytko and Naomi E. Rance

Departments of Pathology, Cell Biology and Anatomy, and Neurology (S.J.K., T.W.A., N.E.R.), University of Arizona College of Medicine, Tucson, Arizona 85724; and Department of Pathology, Section of Comparative Medicine (M.L.V.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Address all correspondence and requests for reprints to: Naomi E. Rance, M.D., Ph.D., Department of Pathology, University of Arizona College of Medicine, 1501 North Campbell Avenue, Tucson, Arizona 85724. E-mail: nrance{at}u.arizona.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we compared the morphology and distribution of neurons expressing GnRH gene transcripts in the hypothalamus and forebrain of the cynomolgus monkey to that of the human. As in the human, three subtypes of GnRH neurons were identified. Type I GnRH neurons were small, oval cells with high levels of gene expression and were located within the basal hypothalamus. Type II GnRH neurons were small and sparsely labeled and were widely scattered in the hypothalamus, midline nuclei of the thalamus, and extended amygdala. Type III neurons displayed magnocellular morphology and intermediate labeling intensity and were located in the nucleus basalis of Meynert, caudate, and amygdala. In a second experiment, we determined the effect of estrogen or estrogen plus progesterone on the gene expression of GnRH neurons in the brains of young, ovariectomized cynomolgus monkeys. We report that hormone treatment resulted in a significant decrease in GnRH mRNA in type I neurons within the basal hypothalamus of ovariectomized monkeys. In contrast, there was no effect of hormone treatment on the gene expression of type III GnRH neurons in the nucleus basalis of Meynert. The present findings provide evidence that the increase in gene expression of type I GnRH neurons in postmenopausal women is secondary to the ovarian failure of menopause. The differential responses of type I and III GnRH neurons to hormone treatment provide additional evidence that distinct subpopulations of neurons expressing GnRH mRNA exist in the primate hypothalamus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MAMMALIAN REPRODUCTION IS controlled by neurons in the central nervous system that secrete GnRH (1). Although the structure of the GnRH decapeptide was elucidated in 1971 (2, 3), our understanding of the mechanisms controlling the synthesis and secretion of GnRH is still limited. This is due, in part, to the diffuse distribution of GnRH neurons that makes them difficult to study (4). In addition, multiple forms of GnRH exist within a single species, including the human (5, 6). The control of GnRH secretion also involves complex feedback circuits that regulate both biological rhythms and monthly reproductive cycles in the female (7). Finally, multiple afferent systems converge on GnRH neurons producing a dynamic, finely tuned reproductive control system (8).

In the human brain, three morphological subtypes of neurons expressing GnRH mRNA were identified using in situ hybridization (9). A group of small, oval neurons with high levels of gene expression (type I) was present within the medial basal hypothalamus, the putative control center for reproduction in the primate (10). A second population of small, round to oval, lightly labeled GnRH neurons (type II) was identified within the septal-preoptic region, bed nucleus-amygdala continuum, and ventral globus pallidus. Finally, a third population of large, round neurons of intermediate labeling intensity (type III) was located in the magnocellular basal forebrain complex, ventral globus pallidus, and putamen (9).

The presence of subtypes of GnRH neurons in the primate brain has been verified in the rhesus and pigtailed monkeys using both immunocytochemical methods and in situ hybridization (6, 11, 12, 13). In the rhesus monkey, two distinct subtypes of GnRH neurons were identified that migrate into the brain at different times during embryogenesis (11). The molecular form of GnRH expressed in these two subgroups was also different. The early migrating group expressed a fragment of GnRH (amino acids 1 through 5) and a GnRH cleavage enzyme, a metalloendopeptidase. In contrast, the late migrators expressed the fully mature mammalian GnRH decapeptide (6). In the fetal rhesus brain, the early migrating GnRH cells differentiated into two morphological subtypes, corresponding to the type II and type III GnRH mRNA-containing neurons in the human, whereas the late migrators assumed the morphological appearance and distribution of the type I GnRH mRNA-containing neurons (11).

The present study was designed to examine the morphology and topography of neurons expressing GnRH gene transcripts in the hypothalamus and basal forebrain of the cynomolgus monkey and to determine if this species displays subtypes of GnRH neurons similar to the human. We also examined the effect of hormone treatment on the gene expression of GnRH neuronal subtypes in the brains of young, ovariectomized cynomolgus monkeys. We have previously demonstrated that GnRH gene expression is increased in type I neurons within the medial basal hypothalamus of postmenopausal women (14). These data suggested that the ovarian failure of menopause resulted in increased gene expression of type I GnRH neurons in the human hypothalamus, although a confounding effect of age could not be eliminated. To test this hypothesis, we determined the effect of hormone treatment on the gene expression of type I GnRH neurons within the hypothalamus of young, ovariectomized cynomolgus monkeys. The present study also examined the effect of ovarian steroids on the magnocellular (type III) GnRH neurons in the nucleus basalis of Meynert. We focused on the type I and type III neurons because these neurons clearly correspond to the late and early migrating subtypes, respectively, of GnRH neurons as described by Quanbeck et al. (11). Therefore, this study was designed to compare the response of the late (type I) and early (type III) migrating subgroups of GnRH neurons to hormone treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Distribution and morphology of neurons expressing GnRH mRNA in the hypothalamus and basal forebrain of intact cynomolgus monkeys

Four adult female cynomolgus macaques (Macaca fascicularis, 8–10 yr of age) were obtained from Primate Products Inc. (Miami, FL). The animals were individually housed at the Comparative Medicine Clinical Research Center at the Wake Forest University School of Medicine. Animal treatments from both experiments were carried out in compliance with state and federal laws, standards of the Department of Health and Human Services, and the guidelines of the Institutional Animal Care and Use Committee at the Wake Forest University School of Medicine. Menstrual cycles were monitored by visual inspection and daily swabbing. The monkeys were killed during the midfollicular phase. They were restrained with ketamine (15 mg/kg im), deeply anesthestized with sodium pentobarbital (35 mg/kg, iv), and perfused transcardially with cold 0.1 M PBS (pH 7.4). The brains were rapidly removed and sliced into 1-cm slabs with the aid of a monkey brain matrix. Hypothalamic blocks were dissected out, snap frozen, and stored at -80 C. The hypothalamic blocks were serially sectioned (12 µm thickness) in a cryostat. The sections were thaw mounted onto gelatin-coated slides and stored at -80 C until hybridization.

In situ hybridization was performed on every 40th section using a [35S]-labeled 48-base cDNA probe complementary to bases 1128 to 1175 of the human GnRH gene (15). A GenBank search showed no significant homology to other mammalian central nervous system genes, including other molecular forms of the GnRH gene (16). For each study, all sections were processed within the same hybridization procedure as previously described (9). The probe was labeled on the 3' end using terminal deoxynucleotidyl transferase (Invitrogen, Indianapolis, IN) and [35S]deoxyadenosine triphosphate (>1000 Ci/mmol, NEN Life Science Products, Boston, MA). Slides were brought to room temperature, postfixed in 4% formaldehyde in PBS for 5 min, treated with 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl (pH 8.0) for 10 min, and delipidized in a graded series of ethanol and chloroform. After drying, slides were incubated for 20 h at 37 C in 60 µl buffer consisting of 50% formamide, 600 mM NaCl, 80 mM Tris-HCl (pH 7.5), 4 mM EDTA, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 10% dextran sulfate, 0.2 mg/ml heparin sulfate, 100 mM dithiothreitol, and 106 dpm of [35S]-labeled probe. Slides were then washed in a solution of 0.3 M NaCl/30 mM sodium citrate buffer (2x SSC) and 50% formamide at 40 C, followed by washes in SSC at room temperature. The slides were dipped in nuclear emulsion, air dried, and stored at 4 C. Test slides were developed to determine optimal exposure time for visualization of the maximum number of GnRH neurons. The slides were exposed for 2 months and then developed and counterstained with toluidine blue. As a control, monkey hypothalamic sections were also hybridized with a 48-base scrambled GnRH probe directed to the same sequence of the GnRH gene described above. This procedure resulted in the absence of neuronal labeling.

All hybridized sections were scanned manually using a combination of brightfield and darkfield microscopy. There were no qualitative differences in labeling among the four monkeys, and a representative hypothalamus was chosen for computer-assisted mapping. Every hybridized section from this representative monkey was mapped using an image-combining computer microscope equipped with a motorized stage, a Lucivid miniature CRT, and Neurolucida software (Microbrightfield, Baltimore, MD). Sections were systematically scanned in an overlapping sequence with the aid of an Optiphot Microscope (Nikon), a 10X planapo lens (Nikon, Tokyo, Japan), and a darkfield condenser. Labeling of cells was verified using brightfield illumination at x200 magnification. A neuron was considered labeled if the silver grains showed the typical cytoplasmic distribution and numbered five times greater than nonspecific background labeling. Neurons were classified into subtypes according to the criteria described in our previous study of the human hypothalamus (9).

Effects of hormone treatment on the gene expression of type I and III GnRH neurons in the ovariectomized cynomolgus monkey

This experiment was performed using hypothalamic sections obtained from a large study designed to determine the effects of hormone treatment on coronary arteriosclerosis in the primate (17). Sections from these monkeys were also used in an earlier experiment examining the effects of hormone treatment on neurokinin B and proopiomelanocortin gene expression in the primate hypothalamus (18). We studied 24 adult female cynomolgus macaques (5–13 yr of age) that were ovariectomized and, 24 months later, divided into three experimental groups: untreated ovariectomized controls (OVX); continuous estrogen treatment (OVX + E); or estrogen plus progesterone treatment (OVX + EP). Conjugated equine estrogen (Premarin, Wyeth-Ayerst, Radnor, PA) was used for an initial treatment period of 7.2 µg/d for 8 months and then increased to 0.17 mg/d (per 4 kg body weight). Medroxyprogesterone acetate (Cycrin, ESI Lederle, Philadelphia, PA) was given at a dose of 650 µg/d (per 4 kg body weight). The hormones were administered twice daily in the diet. The treatment doses were designed to duplicate those commonly prescribed to postmenopausal women (equivalent to 0.625 mg Premarin and 2.5 mg medroxyprogesterone per day). The three groups of animals received either no hormone treatment or one of the two hormone treatments for a total of 30 months before killing (as described above). For detailed information concerning dosages and plasma levels of hormones, see Refs. 18 and 19 .

The sectioning and processing of the hypothalami from the ovariectomized animals were identical to the intact animals described above. Eight adjacent sections matched to plate 790 of the Bleier monkey brain atlas (20) were hybridized with the GnRH probe. Based on the mapping of GnRH neurons in the intact monkey, this level was determined to provide the best sampling of type I neurons in the basal hypothalamus and type III neurons in the nucleus basalis of Meynert (see Fig. 2BGo). Test sections were developed at 2, 4, and 6 d after dipping into nuclear emulsion. The slides were developed after 8 d of exposure to provide the optimum autoradiographic labeling density for quantitative analyses of type I and III neurons. Sections were counterstained with toluidine blue. Two OVX animals and one OVX + E animal were excluded from this study because of sectioning artifact. No type II GnRH neurons were detected after 8 d of exposure in the OVX and OVX plus hormone-replaced animals (14). Therefore, type II neurons were not included in the quantitative analysis of the effects of hormone replacement on GnRH gene expression. Control sections hybridized with a 48-base scrambled probe directed to the same sequence of the GnRH gene revealed no labeling of neurons.



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  		Figure 2. Computer-assisted maps of neurons containing GnRH mRNA in the hypothalamus and basal forebrain of an intact cynomolgus monkey in the midfollicular phase (sections 20 and 100). The maps in Figs. 2Go and 3Go are plotted at successive anterior to posterior intervals with the section number at the lower right of each map. Each symbol represents one neuron. The filled triangles indicate type I GnRH neurons, the open circles indicate type II neurons, and the filled squares indicate type III neurons. ac, Anterior commissure; AMYG, amygdala; HIP, hippocampus; INF, infundibular nucleus; MTN, midline thalamic nuclei; NBM, nucleus basalis of Meynert; oc, optic chiasm; ot, optic tract; P, putamen; ps, pituitary stalk; PVN, paraventricular nucleus; SON, supraoptic nucleus; VM, ventromedial nucleus; 3V, third ventricle.

 
Slides were coded before analysis to prevent experimenter bias. For each monkey, labeled GnRH neurons were mapped on six consecutive sections using the image-combining computer microscope system and systematic scanning technique as described above. Quantitative analyses of cell profile area and autoradiographic grains per cell were performed on all labeled type I neurons. In the nucleus basalis of Meynert, analysis was performed on 20 randomly selected type III neurons. Images of labeled neurons were captured using Neurolucida software and a CCD-100 camera (Dage-MTI, Michigan City, IN), and they were exported into Simple PCI software (Compix Inc., Cranberry Township, PA) for analysis. The cell perimeters were digitized manually for calculations of cell profile areas. The number of autoradiographic grains per labeled cell was determined by calculating the number of grains per cell and subtracting from this value the background grains measured on an adjacent, unlabeled cell. For statistical analysis, the mean cell profile area and the mean number of grains per cell were calculated for each animal. These means were then used to calculate the mean and SEM of each experimental group. Group means were compared using two-factor ANOVA (treatment x cell type), followed by Tukey’s multiple comparison procedures ({alpha} = 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution and morphology of neurons expressing GnRH mRNA in the hypothalamus and basal forebrain of intact cynomolgus monkeys

Neurons expressing GnRH gene transcripts were widely scattered throughout the hypothalamus and basal forebrain of the intact cynomolgus monkey. As in the human, these neurons could be classified into three distinct subtypes based on their morphological appearance and grain density. The classification scheme of neuronal subtypes expressing GnRH mRNA has been previously presented in detail for the human hypothalamus and basal forebrain (9).

Type I GnRH neurons were round to oval and heavily labeled with autoradiographic grains (Fig. 1AGo). These neurons frequently exhibited a single elongated process. Type I neurons were sparsely scattered in the medial basal hypothalamus and extended laterally within the hypothalamus superior to the optic chiasm and optic tracts (Figs. 2Go and 3Go). Several type I neurons were localized within the supraoptic nucleus. Type I neurons were also seen in the periventricular hypothalamus extending dorsally to include the paraventricular nucleus. Neurons with type I morphology were occasionally identified in the amygdala and hippocampus.



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  		Figure 1. Representative photomicrographs of the subtypes of GnRH neurons from intact cynomolgus monkeys in the midfollicular phase (experiment 1, slides developed after 2 months of exposure). The autoradiographic grains mark the location of GnRH mRNA, and the sections are counterstained with toluidine blue. A, Type I GnRH neuron within the medial basal hypothalamus. B, Type II GnRH neuron within the medial hypothalamus. C, Type III magnocellular GnRH neurons within the nucleus basalis of Meynert. Bar, 10 µm on all photomicrographs.

 


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  		Figure 3. Continuation of computer-assisted maps of neurons containing GnRH mRNA in the hypothalamus and basal forebrain of an intact cynomolgus monkey in the midfollicular phase (sections 180 and 260). See legend to Fig. 2Go for details.

 
Type II GnRH neurons were small to medium in size, round or oval in shape, and sparsely labeled with autoradiographic grains, compared with the type I subtype (Fig. 1BGo). More type II GnRH neurons were identified in the cynomolgus monkey brain than in our previous studies of the human hypothalamus.

Type II GnRH neurons were located within the medial hypothalamus including the ventromedial nucleus (Figs. 2Go and 3Go). These neurons were also scattered within the bed nucleus of the stria terminalis, ventrolaterally through the nucleus basalis to the amygdala (extended amygdala), and in the midline thalamic nuclei.

Type III GnRH neurons were magnocellular in appearance with large nuclei and nucleoli, prominent Nissl substance, and a grain density intermediate between the type I and type II subgroups (Fig. 1CGo). These neurons were scattered within the magnocellular basal forebrain with occasional clusters of four to eight labeled neurons (Figs. 2Go and 3Go). Medium to large GnRH neurons were also identified in the corpus striatum and the amygdala, but these were slightly smaller in size relative to the magnocellular neurons in the nucleus basalis.

Effects of hormone treatment on the gene expression of type I and type III GnRH neurons in the OVX cynomolgus monkey

The distribution and morphology of type I and III GnRH neurons in the OVX, OVX + E, and OVX + EP animals were similar to that of the intact group described above. The 8-d exposure time resulted in grain densities that were considerably less than those seen after 2 months. This exposure time, however, was optimal for quantification of the autoradiographic grains associated with type I or III neurons. As with our previous studies of the human hypothalamus (14), no type II GnRH neurons were detected after only 8 d of exposure.

Quantitative analysis revealed that E or E + P treatment of OVX animals significantly reduced the number of autoradiographic grains associated with each type I GnRH neuron (Fig. 4Go). Two-factor ANOVA revealed a significant overall effect of hormone treatment (F = 7.418; df = 2, 35; P = 0.002). Post hoc comparisons (Tukey’s honestly significant difference tests) showed that hormone treatment significantly reduced the number of autoradiographic grains in type I GnRH neurons (P < 0.05). In contrast, there was no effect of hormone treatment on the autoradiographic grains in type III GnRH neurons. Two-factor ANOVA also revealed a significant overall effect of the neuronal subtype on autoradiographic grain number (F = 133.4; df = 1,35; P < 0.001). Post hoc comparisons revealed that the type I GnRH neurons had significantly more autoradiographic grains than type III neurons across all treatment groups (P < 0.05, Fig. 4Go).



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  		Figure 4. Hormone treatment significantly reduces the number of grains per type I GnRH neuron in the hypothalamus of OVX cynomolgus monkeys (A). In contrast, the number of grains per type III GnRH neuron in the nucleus basalis of Meynert is not significantly different among treatment groups (B). Hormone treatment does not affect the cell profile area of types I or III GnRH neurons (C and D). Type I GnRH neurons displayed greater numbers of autoradiographic grains and were smaller than type III neurons, regardless of treatment group. OVX, ovariectomized; OVX + E, ovariectomized plus estrogen; OVX + EP, ovariectomized plus estrogen and progesterone. Data represent mean ± SEM. *, Significantly different from OVX (Tukey’s honestly significant difference multiple comparison test with P < 0.05).

 
Quantitative measurements of cell profile areas confirmed the striking difference in cell size between the type I and III GnRH neurons. As expected from the morphological appearance of these neurons, the mean profile area of type I neurons was considerably smaller than that of type III neurons (type I: 241.0 ± 67.8 µm2 vs. type III: 907.1 ± 78.9 µm2, mean + SEM, pooled data from all treatments). Two-factor ANOVA showed a significant overall effect of neuronal subtype on mean cell profile area (F = 223.5; df = 1,31; P < 0.001). Post hoc comparisons confirmed that type I neurons were significantly smaller than the type III neurons, regardless of treatment group. Hormone treatment had no effect on the mean cell profile area of either the type I or III GnRH neurons (Fig. 4Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, in situ hybridization and computer-assisted microscopy were used to map the distribution of neurons expressing GnRH mRNA in the hypothalamus and basal forebrain of the cynomolgus monkey. Similar to the descriptions of GnRH neurons in a variety of species (4), these neurons were not confined to a specific brain region but were widely scattered in the basal hypothalamus and forebrain. In addition, based on morphology and gene expression, three subtypes of neurons expressing GnRH mRNA were identified. The appearance and distribution of neurons expressing GnRH gene transcripts in the cynomolgus monkey brain were remarkably similar to those previously observed in the human hypothalamus and basal forebrain (9).

We report that E or E + P decreased GnRH gene expression in the type I neurons in the basal hypothalamus of OVX cynomolgus monkeys. These data provide evidence that the increase in GnRH gene expression in the type I neurons of older women is related to the ovarian failure of menopause (14). In the monkey, the ventral hypothalamic tract was formed by cells with the morphological features and distribution of type I GnRH neurons (21). These neurons project to the primary capillary plexus of the median eminence, suggesting that their function is related to the regulation of gonadotropin secretion from the anterior pituitary gland (22). The present findings support this hypothesis and suggest that type I GnRH neurons are a site of steroid negative feedback in the primate brain.

Early physiological studies of the rhesus monkey suggested that estrogen negative feedback operated primarily at the level of the anterior pituitary gland (23). This conclusion was based on the finding that estrogen suppressed LH secretion in animals with lesions of the medial basal hypothalamus receiving infusion of unvarying pulses of GnRH (24). In addition, initial studies were unable to demonstrate a suppressive effect of estradiol on GnRH secretion in female monkeys when measured by either portal blood collection (25) or push-pull perfusion (26). Although estradiol inhibited LH release when directly implanted into the medial basal hypothalamus (27), a pituitary site of action could not be excluded by these studies because of the close anatomic relationship of the median eminence with the anterior pituitary gland.

More recently, a suppressive effect of estrogen on GnRH secretion in the primate has been demonstrated using push-pull perfusion cannulae implanted in the median eminence (28). Furthermore, the synchronized, multiunit electrical activity in the medial basal hypothalamus that coincides with pulses of LH in peripheral plasma (the GnRH pulse generator) was modified by estrogen replacement (29). Removal of the ovaries led to a prolonged duration and an increase in the maximal frequency of the GnRH pulse generator, and these effects were reversed by treatment with estrogen (29). Finally, recent studies using RNAase protection assays showed increased GnRH mRNA in the basal hypothalamus of the male rhesus in response to orchidectomy (30) and a suppressive effect of estradiol administration on GnRH gene expression in the ovariectomized female monkey (31).

The cellular mechanisms of estrogen feedback on GnRH neurons are currently a subject of active investigation. There is considerable evidence that estrogen acts at multiple sites, including modulation of transsynaptic inputs, glial interactions, or directly on the plasma membrane (8, 32). In addition, estrogen has been shown to regulate synapse formation on GnRH neurons in adult monkeys (33, 34). Although estrogen {alpha}-receptors have not been identified in GnRH neurons, recent studies in laboratory rodents suggests that estrogen ß-receptors are colocalized in GnRH neurons (35, 36, 37, 38). These studies raise the possibility that estrogen also has direct genomic actions on GnRH neurons.

Our mapping study of neurons expressing GnRH mRNA revealed an increased number of type II GnRH neurons in the ventromedial nucleus and midline thalamic nuclei of the intact monkey relative to that of the human (9). Although these findings may represent a species difference, they may also be a consequence of the shorter exposure time of the present study, resulting in a decreased number of background silver grains and better detection of neurons with low levels of gene expression. Neurons expressing GnRH mRNA have also been identified within the ventromedial nucleus of the pigtailed monkey using sensitive RNA probes (13). However, retrograde tracing studies in the cynomolgus monkey have failed to demonstrate GnRH-immunoreactive neurons projecting to the median eminence from the ventromedial nucleus (22). In addition, numerous studies of the rat hypothalamus have not revealed significant projections of the ventromedial nucleus to pituitary portal system (39, 40, 41). Therefore, it seems unlikely that the ventromedial type II GnRH neurons participate directly in the regulation of LH secretion.

In contrast with the decrease of GnRH gene transcripts in type I neurons, there was no effect of hormone treatment on the expression of GnRH mRNA in the magnocellular (type III) neurons in the nucleus basalis of Meynert. This subtype of GnRH neuron was first characterized in the human basal forebrain with the use of in situ hybridization (9). Although the existence of magnocellular GnRH neurons was initially controversial, their presence has been subsequently confirmed in both the rhesus and pigtailed macaque using both immunocytochemistry and in situ hybridization (6, 11, 12, 13). The truncated form of the GnRH peptide in the magnocellular neurons explains their limited reactivity to most antisera and the failure of previous immunocytochemical studies to identify these neurons in the primate brain (6, 11). The present study shows that the type I and III GnRH neurons respond differentially to ovarian hormone treatment. The absence of a response by the magnocellular (type III, early migrating) GnRH neurons to ovarian steroids, combined with their location in the nucleus basalis, suggests that these neurons do not participate in the regulation of LH secretion. These data add to a growing body of evidence that different forms of the GnRH peptide may have novel functions that are unrelated to release of gonadotropins (42).


   Acknowledgments
 
We gratefully acknowledge the donation of cynomolgus monkey hypothalami by Dr. Thomas Clarkson at the Comparative Medicine Clinical Research Center at the Wake Forest University School of Medicine. We thank Nathaniel McMullen, Tatiana Sandoval-Guzman, Carla Escobar, and Seth Stalcup for useful comments on an earlier version of this manuscript.


   Footnotes
 
Present address for T.W.A.: Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, Maryland.

This work was supported by NIH/National Institute on Aging Grant AG-09214.

Abbreviations: OVX, Ovariectomized controls; OVX + E, continuous estrogen treatment; OVX + EP, estrogen plus progesterone treatment.

Received June 7, 2002.

Accepted October 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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